ABSTRACT ? Project 2 Triple negative breast cancer (TNBC) represents an aggressive subtype of breast cancer, characterized by significant intratumor heterogeneity, limited treatment options, and poor patient outcome. The inability to effectively treat TNBC is thought to be in part due to its heterogeneity, as cells are highly plastic and able to respond rapidly to therapeutic insults to steer into drug resistant states. One aspect that is likely to strongly influence TNBC plasticity, heterogeneity, and response to therapy is the microenvironment (ME) in which cells reside. Interactions with extracellular matrix proteins or soluble factors like growth factors and cytokines can profoundly change phenotypic properties of TNBC cells, and mounting evidence suggests that such ME factors also influence response to therapy. We hypothesize that the ME impacts therapeutic response of TNBC, and that consideration of signals from the ME in treatment decisions are likely to lead to improved therapeutic control and patient outcomes. We propose to couple experimental assessment of TNBC response to targeted therapeutics in the presence of defined combinatorial ME perturbations (MEPs) with concomitant expression profiling and computational approaches to define underlying pathway signatures to identify vulnerabilities in residual cancer cells that could be exploited for therapeutic benefit. This will be accomplished in three Aims. In Aim 1, we will utilize a novel technology known as microenvironment microarrays (MEMA), which allow for the rational interrogation of thousands of unique ME for effects on cellular phenotypes in a single assay, to identify MEPs that confer resistance to six targeted therapeutics in TNBC cell lines and primary patient derived xenograft (PDX) samples. In Aim 2, we will perform expression profiling by RNA-Seq at fixed time points on TNBC cells grown in the presence of resistance conferring MEPs plus therapeutic and use computational approaches to identify underlying reduced dimensionality network signatures (PREdic-tors of CEllular Phenotypes to guide Therapeutic Strategies, PRECEPTS) that are altered as a result of interactions of cells with MEP and drug. These altered PRECEPTS signatures represent candidates for therapeutic intervention, and will be tested using drug combinations in an attempt to overcome ME-mediated resistance. In Aim 3, we will perform dynamic imaging and expression profiling of the response of TNBC cells to resistance conferring MEPs plus drug and identify PRECEPTS signatures that are dynamically altered. Such PRECEPTS signatures represent potential transition vulnerabilities that could be targeted for therapeutic intervention, which we will test experimentally using drug combination treatments of TNBC cells. These approaches will be closely coordinated with those of Projects 1 and 3 in the use of common cell lines, drugs, and reagents and to maximize the information that we derive from the experiments. This approach should enable the discovery of new drug combinations that could be deployed clinically to improve outcome in TNBC patients with primary and disseminated disease.